Synthesis,characterization and immunochemical evaluation of cephalosporin antigenic determinants

Lack of knowledge of the exact chemical structure of cephalosporin antigenic determinants has hindered clinical interpretation of adverse reactions to these drugs and delayed understanding of the mechanisms involved in the specific recognition and binding of IgE molecules to these antigenic determinants. We further resolve the relationship between structure and activity of proposed antigenic chemicals, including the rational design and synthesis of these haptenic structures. Comparative RAST inhibition studies of the synthesized molecules revealed that they were recognized by IgE antibodies induced by cephalosporin antibiotics. Thus, these data indicate that recognition is mainly directed to the acyl side chain and to the beta-lactam fragment that remains linked to the carrier protein in the cephalosporin conjugation course.     

Opposite effects of galectin-1 on alternative metabolic pathways of L-arginine in resident,inflammatory, and activated macrophages

Recent evidence has implicated galectins and their carbohydrate ligands as master regulators of the inflammatory response. Galectin-1, a member of this family, has shown specific anti-inflammatory and immunoregulatory effects. To gain insight into the potential mechanisms involved in these effects, we investigated the effects of galectin-1 in L-arginine metabolism of peritoneal rat macrophages. Pretreatment of macrophages with galectin-1 resulted in a dose- and time-dependent inhibition of lipopolysaccharide-induced nitric oxide (NO) production, accompanied by a decrease in inducible nitric oxide synthase (iNOS) expression (the classic pathway of L-arginine). On the other hand, galectin-1 favored the balance toward activation of L-arginase, the alternative metabolic pathway of L-arginine. Inhibition of NO production was not the result of increased macrophage apoptosis because addition of this beta-galactoside-binding protein to macrophages under the same experimental conditions did not affect the apoptotic threshold of these cells. To understand how endogenous galectin-1 is regulated in macrophages under inflammatory stress, we finally explored the ultrastructural distribution, expression, and secretion of galectin-1 in resident, inflammatory, and activated macrophages. This study provides an alternative cellular mechanism based on the modulation of L-arginine metabolism to understand the molecular basis of the anti-inflammatory properties displayed by this carbohydrate-binding protein.     

Photosynthetic acclimation to photon irradiance and its relation to chlorophyll fluorescence and carbon assimilation in the halotolerant green alga Dunaliella viridis

This work describes the long-term acclimation of the halotolerant microalga Dunaliella viridis to different photon irradiance, ranging from darkness to 1500 μmol m⁻² s⁻¹. In order to assess the effects of long-term photoinhibition, changes in oxygen production rate, pigment composition, xanthophyll cycle and in vivo chlorophyll fluorescence using the saturating pulse method were measured. Growth rate was maximal at intermediate irradiance (250 and 700 μmol m⁻² s⁻¹). The increase in growth irradiance from 700 to 1500 μmol m⁻² s⁻¹ did not lead to further significant changes in pigment composition or EPS, indicating saturation in the pigment response to high light. Changes in Photosystem II optimum quantum yield (F_v/F_m) evidenced photoinhibition at 700 and especially at 1500 μmol m⁻² s⁻¹. The relation between photosynthetic electron flow rate and photosyntetic O₂ evolution was linear for cultures in darkness shifting to curvilinear as growth irradiance increased, suggesting the interference of the energy dissipation processes in oxygen evolution. Carbon assimilation efficiencies were studied in relation to changes in growth rate, internal carbon and nitrogen composition, and organic carbon released to the external medium. All illuminated cultures showed a high capability to maintain a C:N ratio between 6 and 7. The percentage of organic carbon released to the external medium increased to its maximum under high irradiance (1500 μmol m⁻² s⁻¹). These results suggest that the release of organic carbon could act as a secondary dissipation process when the xanthophyll cycle is saturated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Construction and characterization of BAC libraries for three fish species; rainbow trout, carp and tilapia

Bacterial artificial chromosome (BAC) libraries are important tools for genomic research. We have constructed seven genomic BAC libraries from three fish species, rainbow trout (Oncorhynchus mykiss), carp (Cyprinus carpio) and tilapia (Oreochromis niloticus). The two rainbow trout BAC libraries have average insert sizes of 58 and 110 kb. The average size of inserts in the carp BAC library is 160 kb. The average insert sizes of the four tilapia BAC libraries are 65, 105, 145 and 194 kb, respectively. These libraries represent good coverage of each genome (2-64 x coverage). The libraries can be screened by conventional colony hybridization and provide a starting point for the construction of high-density filtres or polymerase chain reaction (PCR) screening approaches. These BAC libraries will facilitate the positional cloning of quantitative trait loci (QTLs) for a variety of economically important traits in these species.     

Further Characterization of 5-HT1A Receptors in the Goldfish Retina: Role of Cyclic AMP in the Regulation of the In Vitro Outgrowth of Retinal Explants

The presence of serotonin 5-HT_1A receptors and their physiological role were further characterized in the goldfish retina. The effects of the 5-HT_6/7 receptor antagonists pimozide, fluphenazine and amoxapine, the 5-HT_1A receptor antagonist WAY-100,135, and the alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, on the 5-HT_1A receptor agonist [³H]8-hydroxy-2-(di-n-propylamino)tetralin binding to retinal membranes, were evaluated. In addition, the effects of serotonin, 8-hydroxy-2-(di-n-propylamino)tetralin, WAY-100,135, the adenylate cyclase inhibitors SQ22536 and MDL12330A, and the cyclic AMP analog 8-bromoadenosine-3:5 cyclic monophosphate were also studied on neuritic outgrowth from retinal explants. WAY-100,135 but not 5-HT_6/7receptor antagonists inhibited [³H]8-hydroxy-2-(di-n-propylamino)tetralin binding to retinal membranes N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline decreased [³H]8-hydroxy-2-(di-n-propylamino)tetralin binding sites up to 70%, while receptor turnover was similar to that reported in other tissues. Serotonin and 8-hydroxy-2-(di-n-propylamino)tetralin stimulated cyclic AMP production, both ex vivo and in vitro, and these increases were related to inhibition of neuritic outgrowth. The inhibitory effect was reduced by SQ22536 and by WAY-100,135, and was mimicked by 8-bromoadenosine-3:5cyclic monophosphate. This study supports previous findings about the role of serotonin as a regulator of axonal outgrowth during in vitro regeneration of the goldfish retina and demonstrates that this effect is mediated, at least in part, by 5-HT_1A receptors through a mechanism which involves an increase of cyclic AMP levels.     

Localization of gamma-tubulin to the basal foot associated with the basal body extending a cilium

The human oviduct epithelium primarily consists of ciliated cells and secretory cells. Solitary cilia usually extend from the apical surface of the secretory cells. We investigated the localization of -tubulin in the ciliary basal apparatus of both cell types by fluorescence immunohistochemistry and immunoelectron microscopy. In addition to basal bodies, -tubulin was identified in the lateral basal foot, especially the basal foot cap. This observation is consistent with previous observations that microtubules radiate from the basal foot and the basal foot serves as the microtubule organizing centre.     

Roles of His-79 and Tyr-180 of D-xylose/dihydrodiol dehydrogenase in catalytic function

Mammalian dimeric dihydrodiol dehydrogenase is identical with d-xylose dehydrogenase and belongs to a protein family with prokaryotic proteins including glucose-fructose oxidoreductase. Of the conserved residues in this family, either His-79 or Tyr-180 of d-xylose/dihydrodiol dehydrogenase has been proposed to be involved in the catalytic function. Site-directed mutagenesis was used to examine the roles of the two residues of the monkey enzyme. A mutant, Y180F, was almost inactive, but, similarly to the wild-type enzyme, exhibited high affinity for NADP(H) and fluorescence energy transfer upon binding of NADPH. The H79Q mutation had kinetically largest effects on K(d) (>7-fold increase) and K(m) (>25-fold increase) for NADP(H), and eliminated the fluorescence energy transfer. Interestingly, the dehydrogenase activity of this mutant was potently inhibited with a 190-fold increase in the K(m) for NADP(+) by high ionic strength, which activated the activity of the wild-type enzyme. These results suggest a critical role of Tyr-180 in the catalytic function of this class of enzymes, in addition to functions of His-79 in the coenzyme binding and chemical steps of the reaction.